Inducible IL-33 expression by mast cells is regulated by a calcium-dependent pathway

Abstract

IL-33 is an IL-1 family cytokine that displays dual functions: a cytokine via its receptor, T1/ST2, or a chromatin-binding factor within the nucleus. Functionally, it promotes Th2-associated immunity by enhancing the activation and survival of several cell types. However, the pathways regulating IL-33 expression are still unclear. Although several cells display constitutive expression of IL-33, we showed previously that mast cells expressed low levels of IL-33 constitutively but that IL-33 was induced upon IgE-mediated activation. This was mediated via a calcium-dependent mechanism. In this study, we define the pathway through which this inducible IL-33 is regulated. Importantly, this pathway does not alter expression in cells with high constitutive IL-33 expression, such as epithelial cells or fibroblasts. Our data show that, upstream of calcium, inhibition of PI3K and Sphk activity decreases inducible IL-33 expression to IgE/Ag activation. Additionally, expression of Sphk1 short hairpin RNA prevents upregulation of IL-33 expression. Downstream of calcium, NFAT activity is necessary and sufficient for inducible IL-33 expression. We also demonstrate calcium-dependent transcription from two regions of the IL-33 gene that contain putative NFAT-binding sites, one upstream of exon 1 and one upstream of the start site. Interestingly, we show that blocking other calcium pathways, including inositol triphosphate receptor, or NF-κB inhibits IgE-driven IL-1β, another IL-1 family cytokine, but it has no influence on inducible IL-33 expression. In summary, our data demonstrate cell-specific differences in the regulation of IL-33 expression and define a pathway critical for the expression of inducible IL-33 by mast cells upon their activation.

Figure 1. Ionomycin-induced IL-33 expression is cell-type dependent

A. BMMC, MC/9, NIH3T3, HEVa and CMT93 cells were stimulated with 0.25 μM ionomycin (gray bar) for 4 hours and IL-33 mRNA expression was analyzed by real-time RT-PCR. B. BMMCs with or without 1 μM ionomycin stimulation were intracellularly stained with FITC-anti-IL-33 and mounted with DAPI–containing buffer. Blue, DAPI staining for nucleus. Green, IL-33 staining. Images are representative of 3 independent experiments. Gene expression is pooled from 3 independent experiments, n=3; *p<0.05, ***p<0.001.

Figure 2. IL-33 expression in mast cells is regulated by Sphk-induced calcium mobilization

BMMCs were pretreated with 10μM DMS, or 50μM 2-APB, or both 30 minutes prior to activation by IgE/DNP (black bar). IL-33 (A) and IL-1β (D) gene expression was analyzed by real-time RT-PCR. B. IL-33 expression was inhibited by DMS in a dose-dependent manner. C. IL-33 expression was analyzed in BMMCs pretreated with 10μM DMS and activated with IgE/DNP (black bar) or 0.25μM ionomycin (gray bar). n=3 from 3 independent experiments; *p<0.05, **p<0.01, ***p<0.001.

Figure 3. S1PR-mediated autocrine effects are required for IL-33 gene expression in mast cells

BMMCs were pretreated with different doses of W146, or JTE-013, 30 minutes prior to activation by IgE/DNP. IL-33 gene expression was analyzed by real-time RT-PCR. Data represented 5 individual experiments. n=3; *p<0.05

Figure 4. IL-33 expression in mast cells is dependent on PI3K activity

BMMCs were pretreated with different doses of PI3K inhibitors, LY294002 and wortmannin, 30 minutes prior to IgE/DNP (A and C, black bar) and 0.25μM ionomycin (C, gray bar) activation. IL-33 expression was analyzed by real-time PCR. (B) BMMCs pretreated with 10μM LY294002 or 100nM Wortmannin were activated with IgE/DNP for 24 hours. IL-33 protein expression was detected by intracellular staining. Gray line, isotype control; black dot, unstimulated; gray fill, IgE/DNP stimulation; black line, LY294002 treatment; black dash, wortmannin treatment. n=3 from 2-3 individual experiments; *p<0.05, **p<0.01, ***p<0.001, n.s. = no significant difference.

Figure 5. Sphk1 shRNA inhibited IL-33 upregulation upon IgE/DNP activation

BMMCs were transfected with scrambled (top), Sphk1 (middle) or Sphk2 (bottom) shRNA plasmids that also express GFP for 7 days and activated with IgE/DNP. Cells transfected with shRNA plasmids were gated on GFP⁺ cells. IL-33 protein expression of shRNA transfected cells (GFP⁺ gated) was analyzed by intracellular staining 24 hours after stimulation. Data is representative of 4 independent experiments.

Figure 6. IL-33 expression in mast cells is dependent on transcription factor NFAT

BMMCs pretreated with different doses of INCA-6 or CsA are activated with IgE/DNP (A, black bar) or 0.25 μM ionomycin (B, gray bar) for 4 hours. IL-33 gene expression was analyzed by real-time PCR. C. BMMCs were transfected with constitutively active NFATc1 mutant (caNFATc1). Gray line, caNFATc1^-; gray fill, caNFATc1⁺, based on GFP expression. Data represented 2–3 individual experiments. n=3; *p<0.05, **p<0.01, ***p<0.001.

Figure 7. NF-κB is dispensable for IL-33 expression in mast cells

BMMCs were pretreated with different doses of NF-κB inhibitor, BAY 11-7085, and activated with IgE/DNP for 4 hours. IL-33 (A) and IL-1β (B) gene expression were analyzed by real-time PCR. n=3 from 2 independent experiments; **p<0.01, ***p<0.001, n.s. = no significant difference.

Figure 8. Two regulatory regions of il33 support calcium-dependent transcription

The region between −1600 to −1 of il33 upstream according to exon 1 (il-33-exon) and the region between −1000 to +100 within il33 intron 1 according to ATG transcriptional start site (il-33-ATG) were cloned into pGL.3-basic luciferase reporter plasmid. BMMCs were transfected with pGL.3-basic (mock), il-33-exon (A), or il-33-ATG (B) together with pGL.3-RNL as a transfection efficiency control. Cells were activated with 0.25μM ionomycin 24 hours in the presence of vehicle, 10μM INCA-6 or 1.5μM CsA after transfection. Luciferase activity was analyzed 24 hours after stimulation.. Data represented 4–5 independent experiments. **p<0.01, ***p<0.001.

Figure 9. Pathway for inducible expression of IL-33 in mast cells

IL-33 expression in mast cells upon cross-linking of IgE receptors is regulated by a PI3K-Sphk1-S1P-NFAT pathway. NF-κB is dispensable for the expression of IL-33 and, instead regulates IL-1β. S1P receptor also participates in regulating IL-33 expression.